Angiogenesis is a process of growing new blood vessels from existing blood vessels. Most adult vascular system is quiescence, angiogenesis only occurs in some physiological and pathological mechanisms, such as tumor, diabetic retinopathies, arthritis, anemia organs, endometrial hyperplasia, etc. Angiogenesis plays key roles in rapid growth of tumor cells during tumor development (Hanahan and Folkman: Patterns and emerging mechanisms of the angiogenic switch during tumorigenesis, Cell, 1996, 86:353-364). Studies of animal cancer models and human clinical trials have already proved that inhibition of tumor angiogenesis could effectively inhibit tumor growth and development, therefore prolong patient's life. Angiogenesis is mediated and regulated by many biological factors. Main cells mediating angiogenesis are vascular endothelial cells that form the inside wall of blood vessels. Various growth factors can bind to relevant receptors on the surface of vascular endothelial cells, regulate cellular processes via intracellular signal transduction, and therefore mediate angiogenesis.
Among various growth factors, VEGF (vascular endothelial cell growth factor) is the most important angiogenesis factor (Ferrara: VEGF and the quest for tumor angiogenesis factor, Nat. Rev. Cancer, 2002, 10: 795-803; Ferrara: Role of vascular endothelial growth factor in physiologic and pathologic angiogenesis: therapeutic implications, Semin. Oncol., 2002, 29 (6 suppl): 10-14). VEGF could be secreted by many types of cells, but often over-expressed in tumor cells. VEGF functions by binding to appropriate receptors. There are mainly two kinds of VEGF receptors: FLT-1 (fms-like tyrosine kinase) and KDR. In terms of molecular structures, these two receptors both consist of three different functional regions: the first region is the extracellular region, consisting of seven immunoglobulin-like (Ig-like) domains (d1-d7), which has specific affinity to VEGF, and is the key region for binding VEGF; the second region is the trans-membrane region containing hydrophobic amino acid residues; the third region is the intracellular domain that contains tyrosine kinase functioning group, which gets phosphorylated after the receptor is activated by VEGF, triggering the intracellular signal transduction, leading to functional effects of endothelial cells and angiogenesis.
FLT-1 and KDR are mainly distributed in vascular endothelial cells. Thus, VEGF's mediating activity to vascular endothelial cells is highly specific. VEGF promotes endothelial cell differentiations, guides endothelial cell migrations, inhibits apoptosis, induces vascular morphological changes, and is a highly effective pro-angiogenesis factor.
The expression level of VEGF in tumor tissues is higher than that in the normal tissues. In addition, rapid growth of tumor cells often leads to hypoxia inside the tumor, and hypoxia further induces expression of VEGF. Thus, VEGF is the key factor promoting tumor angiogenesis. Many animal studies have shown that inhibiting binding of VEGF to its receptors could effectively inhibit tumor angiogenesis, and therefore inhibit tumor growth. In other angiogenesis-related diseases, such as diabetic retinopathies and arthritis, etc, VEGF is also closely involved in the development of these diseases (Ferrara: Role of vascular endothelial growth factor in physiologic and pathologic angiogenesis: therapeutic implications. Semin. Oncol. 2002, 29 (6 suppl): 10-14).
Because of the critical roles of VEGF in cancers and other diseases, proteins or chemicals that specifically inhibit VEGF have therapeutic potentials. For example, studies have shown that neutralizing antibody against VEGF could effectively inhibit tumor growth (Jain: Tumor angiogenesis and accessibility: role of vascular endothelial growth factor, Semin. Oncol., 2002, 29 (6 suppl): 3-9). Therefore, developing novel effective VEGF inhibitors is important in clinical research. Since FLT-1 and KDR are natural binders of VEGF, there were studies that investigated the anti-angiogenesis roles of the soluble FLT-1 (the extracellular domain of FLT-1) and the soluble KDR (the extracellular domain of KDR) (Yoko Hasumi: Soluble FLT-1 Expression Suppresses Carcinomatous Ascites in Nude Mice Bearing Ovarian Cancer. Cancer Research 62, 2002: 2019-2023). The soluble FLT-1 could effectively inhibit growth of vascular endothelial cells in vitro, but it has a short serum half-life and can not reach effective serum concentration. Similarly, the soluble KDR was also able to inhibit growth of vascular endothelial cells in vitro, but its anti-tumor activity in animal models was not satisfactory (Yoko Hasumi: Soluble FLT-1 Expression Suppresses Carcinomatous Ascites in Nude Mice Bearing Ovarian Cancer. Cancer Research 62, 2002: 2019-2023).
To overcome the shortcomings of the prior art, the present invention provides novel chimeric proteins containing different fragments of FLT-1 and KDR to effectively block the biological activity of VEGF and inhibit angiogenesis.